The present invention relates to the identification of the TRANCE signal transduction pathway in osteoclasts. This pathway can be exploited for high throughput screening of compounds to identify TRANCE modulators (agonists and antagonists). The invention also relates to identification of a specific pathway in osteoclast survival, and permits manipulation of this pathway, which can provide strategies for treating osteoclast-related diseases such as osteoporosis and osteopetrosis.
Members of the tumor necrosis factor (TNF) family regulate an array of cellular responses including survival, activation, differentiation, and apoptosis. TNF-related activation-induced cytokine (TRANCE; (Wong et al., J. Biol. Chem., 272, 25190-25194, 1997) also called receptor-activator of NF-xcexaB (RANK) ligand (RANKL), osteoclast differentiation factor (ODF) and osteoprotegerin (OPG) ligand (OPGL)) is expressed on T cell-receptor activated T cells and stimulates the TRANCE receptor (TRANCE-R/RANK) on dendritic cells to promote their survival and induce the expression of T-cell regulatory cytokines (Wong et al., supra, 1997; Wong et al., J. Exp. Med., 186:2075-2080, 1997; Josien et al., J. Immunol., 162:2562-2568, 1999). Priming of T cells is dependent on dendritic cell activation (Bachmann et al., J. Exp. Med.: 189, 1025-1031, 1999) and TRANCE appears to be critical for T-helper cell responses to certain viral infections (Bachmann, et al., supra, 1999), suggesting that TRANCE regulates dendritic cell function in vivo. Moreover, it has been shown that TRANCE regulates osteoclasts, cells of monocyte/macrophage origin which resorb calcified bone matrix, a process essential in bone development and homeostasis (Lacey, et al., Cell, 93:165-176, 1998; Yasuda, et al., Proc. Natl. Acad. Sci. U.S.A., 95::3597-3602, 1998). TRANCE is expressed on osteoblasts in response to calciotropic hormones and cytokines, and is critical for osteoclast differentiation from hematopoietic precursors (Lacey et al., supra, 1998; Yasuda, et al., supra, 1998). In mature osteoclasts, TRANCE stimulation induces cell spreading and enhances cell survival and bone resorption (Fuller et al., J. Exp. Med., 188:997-1001, 1998).
Mice with a targeted deletion in the TRANCE gene exhibit osteopetrosis, or thickening of bony matrix, due to a defect in osteoclast differentiation (Kong et al., Nature, 397:315-323, 1999). Mice with a targeted deletion of the non-receptor protein tyrosine kinase (PTK), c-Src (Soriano et al., Cell, 64:693-702, 1991) and mice doubly deficient in nuclear factor-kappa B (NF-xcexaB)1 and NF-xcexaB2 (Iotsova, et al., Nat. Med., 3:1285-9, 1997) also exhibit osteopetrosis. However, while osteoclasts fail to differentiate in NF-xcexaB1 and NF-xcexaB2 deficient mice, osteoclasts from c-Src-deficient mice develop normally but are unable to resorb bone. TRANCE is critical for osteoclast maturation and osteoclast activation (i.e., bone resorption, cytoskeletal rearrangements), suggesting a link between TRANCE, NF-xcexaB and c-Src-mediated signaling. In contrast to its role in osteoclastogenesis, TRANCE does not appear to be important for dendritic cell differentiation since mature dendritic cells are detected at normal levels in TRANCE-deficient mice (Kong et al, supra, 1999).
Two receptors for TRANCE have been identified: OPG/OCIF, a secreted decoy receptor (Yasuda, et al., supra, 1998; Simonet et al., Cell, 89:309-319, 1997), and TRANCE-R (Wong et al., supra, 1997), identified as RANK (Anderson et al., Nature, 390:175-179, 1997), an integral membrane protein with an intracellular signaling domain. TRANCE-R, similar to other TNFR family members, lacks intrinsic catalytic activity and interacts with TNF receptor-associated factors (TRAFs) that act as adapters to activate downstream signaling pathways (Rothe et al., Cell, 78:681-692, 1994). TRANCE-R can interact with TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6 when overexpressed in cell lines. TRAF1, TRAF2, TRAF3, and TRAF5 interact with a canonical PXQXT motif in the C-terminal region of the TRANCE-R cytoplasmic tail whereas TRAF6 binds to a unique motif more proximal to the transmembrane domain (Wong et al., J. Biol. Chem., 273:28355-28359, 1998; Galibert et al., J. Biol. Chem., 273:34120-34127, 1998; Darnay et al., J. Biol. Chem., 274:7724-7731, 1999). Several reports demonstrated that while TRAF2appears important for c-Jun N-terminal kinase activation, TRAF6 is most likely the critical mediator of NF-xcexaB activation by the TRANCE-R since TRAF6 -interacting regions of the cytoplasmic tail are necessary and sufficient for NF-xcexaB activation (Wong et al., supra, 1998; Galibert et al., supra, 1998; Darnay et al., supra, 1999). TRAF6 also mediates NF-xcexaB activation by interleukin-1R (Cao et al., Nature, 383:443-446, 1996; Medzhitov et al., Mol. Cell, 2:253-258, 1998), and toll-like receptors (Zhang et al., J. Biol. Chem., 274:7611-7614, 1999), indicating that TRAF6 links several families of cytokine receptors to NF-xcexaB activation.
TRAF family members are prototypically composed of an N-terminal regulatory domain and a conserved C-terminal TRAF domain (Rothe et al., supra, 1994). The TRAF domain mediates oligomerization, interaction with the receptor, and interaction with various signaling effectors while the N-terminal domain contains a RING finger and several zinc fingers which appears to regulate the activity of its associated signaling molecules (reviewed in Arche, et al., Genes Dev., 12:2821-2830, 1998). Several TRAF family members associate with and activate apoptosis signal-regulating kinase 1 (ASK1) which activates c-Jun N-terminal kinase (JNK) (Nishitoh et al., Mol. Cell, 2:389-395, 1998), and also NF-xcexaB inducing kinase (NIK) which induces NF-xcexaB activation (Malinin et al., Nature, 385:540-544, 1997). Although the TRAF domain alone is sufficient to mediate interaction with these kinases, it is not sufficient to induce their activation. The N-terminal RING and zinc fingers of TRAF proteins are also required to intiate downstream signaling although the precise mechanisms by which these regulatory elements function are not understood (Rothe et al., supra, 1995; Malinin, et al., supra, 1998).
Despite the rapid advancement of knowledge of the effects of TNF-related cytokines on cells, there is still a great amount of information about these factors that remains unknown. Thus, there is a need in the art for identification of elements of the TRANCE signal transduction pathway.
There is a further need in the art for identification of elements of the pathway that are involved in signal transduction of other TNF-related cytokines.
These and other needs in the art are addressed by the present invention.